Anti-hypertensive agents

ABSTRACT

Novel compounds are disclosed as potent inhibitors of angiotensin converting enzyme and as orally effective antihypertensive agents. The compounds have the general formula: ##STR1##

BACKGROUND OF THE INVENTION

Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafterreferred to as ACE) occupies a central role in the physiology ofhypertension. The enzyme is capable of converting the decapeptideangiotensin I, having the sequence

    AspArgValTyrIleHisProPheHisLeu

to an octapeptide, angiotensin II by removal of the carboxy-terminalHisLeu. The symbols for various chemical entities are explained in thefollowing table:

Ala=L-alanine

Arg=L-arginine

Asp=L-aspartic acid

<Glu=pyro-L-glutamic acid

Gly=glycine

Hip=Hippuric acid (Benzoyl glycine)

His=L-histidine

Ile=L-isoleucine

Leu=L-leucine

Phe=L-phenylalanine

ΔPro=L-proline

Pro=L-3,4-dehydroproline

Ser=L-serine

Trp=L-tryptophan

Tyr=L-tyrosine

Val=L-valine

ACE=Aniotensin converting enzymeHepes=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

Angiotensin I is formed by the action of the enzyme renin, anendopeptidase found in kidney, other tissues and plasma, acting on aserum α-2 globulin.

Blood pressure is affected by certain peptides found in the blood. Oneof these, angiotensin II, is a powerful pressor (blood pressureelevating) agent. Another, bradykinin, a nonapeptide with the sequenceArgProProGlyPheSerProPheArg is a powerful depressor (blood pressurelowering) agent. In addition to a direct pressor effect, angiotensin IIstimulates release of aldosterone which tends to elevate blood pressureby causing retention of extracellular salt and fluids. Angiotensin II isfound in measurable amount in the blood of normal humans. However, it isfound at elevated concentrations in the blood of patients with renalhypertension.

The level of ACE activity is ordinarily in excess, in both normal andhypertensive humans, of the amount needed to maintain observed levels ofangiotensin II. However, it has been found that significant bloodpressure lowering is achieved in hypertensive patients by treatment withACE inhibitors. [Gavras, I., et al., New Engl. J. Med. 291, 817 (1974)].

ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of thepenultimate peptide bond at the C-terminal end of a variety of acylatedtripeptides and larger polypeptides having an unblocked α-carboxylgroup. The action of ACE results in hydrolytic cleavage of thepenultimate peptide bond from the carboxyl-terminal end yielding asreaction products a dipeptide and a remnant.

The reactivity of the enzyme varies markedly depending on the substrate.At least one type of peptide bond, having the nitrogen supplied byproline, is not hydrolyzed at all. The apparent Michaelis constant (Km)varies from substrate to substrate over several orders of magnitude. Forgeneral discussion of the kinetic parameters of enzyme catalyzedreactions, see Lehninger, A., Biochemistry, Worth Publishers, Inc., NewYork, 1970, pp. 153-157. Many peptides which are called inhibitors ofthe enzymatic conversion of angiotensin I to angiotensin II are in factsubstrates having a lower Km than angiotensin I. Such peptides are moreproperly termed competitive substrates. Examples of competitivesubstrates include bradykinin, and the peptide BPP_(5a) (also calledSQ20475) from snake venom, whose sequence is <GluLysTrpAlaPro.

Numerous synthetic peptide derivatives have been shown to be ACEinhibitors by Ondetti, et al. in U.S. Pat. No. 3,832,337 issued Aug. 27,1974.

The role of ACE in the pathogenesis of hypertension has prompted asearch for inhibitors of the enzyme that could act as antihypertensivedrugs. See for example U.S. Pat. Nos. 3,891,616, 3,947,575, 4,052,511and 4,053,651. A highly effective inhibitor, with high biologicalactivity when orally administered, isD-3-mercapto-2-methylpropanoyl-L-proline, designated SQ14225, disclosedin U.S. Pat. No. 4,046,889 to Ondetti et al., issued Sept. 6, 1977, andin scientific articles by Cushman, D. W. et al., Biochemistry 16, 5484(1977), and by Ondetti, M. et al., Science 196, 441 (1977). Theinhibitor SQ14225 reportedly has an I₅₀ value of 2.3×10⁻⁸ M. The I₅₀value reported by Cushman, et al, supra is the concentration ofinhibitor required to produce 50% inhibition of the enzyme under astandard assay system containing substrate at a level substantiallyabove K_(m). It will be understood that I₅₀ values are directlycomparable when all potential factors affecting the reaction are keptconstant. These factors include the source of enzyme, its purity, thesubstrate used and its concentration, and the composition of the assaybuffer. All I₅₀ data reported herein have been performed with the sameassay system and same enzyme (human urinary ACE) and with anapproximately 1/2 K_(m) level of substrate and are therefore internallyconsistent. Discrepancies with data obtained by other workers may beobserved. Indeed such discrepancies do exist in the literature, forunknown reasons. See, for example, the I₅₀ values for BPP_(9a) reportedby Cushman, D. W., et al., Experientia 29, 1032 (1973) and by Dorer, F.E., et al., Biochim. Biophys. Acta 429, 220 (1976).

The mode of action of SQ 14,225 has been based upon a model of theactive site of ACE developed by analogy with the better known relatedenzyme, carboxypeptidase A. The active site was postulated to have acationic site for binding the carboxyl end group of the substrate and apocket or cleft capable of binding the side chain of the C-terminalamino acid and providing especially tight binding for the heterocyclicring of a terminal proline residue. A similar pocket for the penultimateamino acid residue was postulated, and the published data suggested arather stringent steric requirement, since the D-form of the inhibitorwas substantially more potent than its stereoisomer or the 3-methyl andunsubstituted analogs. The sulfhydryl group on the inhibitor, postulatedto be bound at the active site near the catalytic center, was believedto play a central role in inactivation of the enzyme by combining withthe zinc moiety known to be essential for catalytic activity.Substituents on the sulfhydryl, such as a methyl group, and an S-acetylderivative, substantially reduced potency of the inhibitor. See Cushman,D. W., et al., Biochemistry, supra.

In vitro study of the mechanism by which SQ 14,225 and its analogs actto inhibit ACE has been somewhat hampered by the instability of thesemolecules under ambient conditions. For example, it has been observedthat a fresh aqueous solution of concentration, e.g., 1 mg per ml of SQ14,225 at a pH of about 8 becomes substantially less active uponstanding for as little as 30 minutes, and that activity continues todecrease as the solution stands for longer periods. It is believed thatthis loss in activity is mainly the result of dimerization of SQ 14,225occurring at the sulfhydryl end groups, whereby a disulfide is formedwhich is largely inactive as an inhibitor. Since the free sulfhydrylgroup is highly reactive and may be readily oxidized to polar acidicmoieties such as sulfone and sulfoxide groups, it may also be that theobserved in vitro loss of activity of aqueous solutions of SQ 14,225 onstanding is in some part a consequence of one or more such oxidationreactions, with formation of a sulfone or sulfoxide which does notfunction effectively as an inhibitor for ACE.

Such reports of SQ 14,225 clinical testing as are currently available,some of which refer to the compound under the name "Captopril", suggestthat the product is sufficiently stable in the normal gastric andintestinal environments of most patients to be an effective inhibitorfor ACE when administered orally. It is not yet clear, however, whetherthere may be a group of patients for which SQ 14,225 is substantiallyineffective. Because of the high reactivity of the free sulfhydrylgroup, SQ 14,225 could readily form mixed disulfides with serum,cellular proteins, peptides or other free sulfhydryl group-containingsubstances in the gastric or intestinal environments, in addition to thepossibility for dimer formation or oxidative degradation reactions. Amixed disulfide with protein may be antigenic and, indeed, occasionalallergic reactions have been clinically observed. See Gavras, et al.,New England J. Med. 298, 991 (1978). Disulfides and oxidativedegradation products of SQ 14,225, if formed, may at best be expected tobe largely ineffective as inhibitors. It may be postulated accordinglythat dose response to SQ 14,225 may vary with conditions ofadministration and among individual patients. Moreover, in at least somepatients, unwanted side effects may occur and maintenance of aneffective concentration of the inhibitor in the body may be difficult tocontrol.

Thiolester compounds generally are thought to be highly reactive in thatthe thiolester linkage is readly hydrolyzable to a sulfhydryl moiety anda carboxylic moiety. Thiolesters are accordingly often used as activeester intermediates for acylation under mild conditions. Such groups as,e.g., acetylthio have been used as blocking groups in the above citedOndetti, et al. patents. Thiolester intermediates are also postulated tooccur in the biosynthesis of cyclic peptides such as tyrocidin orgramicidin S. See Lipmann, F. in Accounts Chem. Res. 6, 361 (1973).

Thiolester compounds having potent ACE inhibitory activity and oraleffectiveness as anti-hypertensive agents have been disclosed incopending applications Ser. No. 116,950, filed Jan. 30, 1980, which is acontinuation of Ser. No. 941,289, filed Sept. 11, 1978 (now abandoned infavor of its pending continuation Ser. No. 116,950, filed Jan. 30, 1980)and Ser. No. 958,180, filed Nov. 6, 1978 (abandoned in favor of itscontinuation Ser. No. 116,951, filed Jan. 30, 1980 which was abandonedin favor of its continuation, Ser. No. 295,589, filed Aug. 24, 1981which was abandoned in favor of its continuation, Ser. No. 524,204,filed Aug. 18, 1983, which was abandoned in favor of its continuation,Ser. No. 680,541, filed Dec. 11, 1984, which was abandoned in favor ofits pending continuation, Ser. No. 850,055, filed Apr. 10, 1986), bothincorporated herein be reference. The previously disclosed compoundsare: N-[3-(benzoylphenylalanylthio)-2-D-methylpropanoyl]-L-proline (I₅₀≈1-4×10⁻⁸ M), N-(2-benzoylphenylalanylthiopropanoyl)-L-proline (I₅₀≈4-7×10⁻⁸ M for racemic compound),N-(3-benzoylphenylalanylthiopropanoyl)-L-proline (I₅₀ ≈7×10⁻⁷ M),N-[3-(benzoylphenylalanylthio)-2-D-methylpropanoyl]-L-3,4-dehydroproline,N-(2-benzoylphenylalanylthiopropanoyl)-L- 3,4-dehydroproline (I₅₀=3×10⁻⁹ M for racemic compound), andN-(3-benzoylphenylalanylthiopropanoyl)-L-3,4-dehydroproline. Unlessnoted otherwise, all amino acids are in their L-forms.

Compounds related to SQ 14,225 have been disclosed by Ondetti, et al.,U.S. Pat. Nos. 4,046,889, 4,052,511, 4,053,651, 4,113,715 and 4,154,840.Of interest are disclosed analogs of SQ 14,225 having the five-memberedheterocyclic ring of proline replaced by a four- or a six-membered ring.The inhibitory potencies of such analogs relative to SQ 14,225 are notdisclosed. Substitution of D-proline for L-proline is reported todrastically reduce inhibitory potency of 3-mercaptopropanoyl amino acids(Cushman, D. W., et al., supra).

SUMMARY OF THE INVENTION

Novel inhibitors of ACE are disclosed which have the general formula##STR2## wherein, R is L-arginyl, L-lysyl or L-pyroglutamyl;

A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl,L-isoleucyl, L-leucyl, L-histidyl, or L-valyl;

R₁ is hydrogen or methyl;

R₂ is L-proline, L-3,4-dehydroproline, D,L-3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine-4-carboxylicacid whose imino group is in imide linkage with the ##STR3## and n is 0or 1 such as when n=0, R₁ is methyl. The disclosed compounds areinhibitors of ACE and are useful as orally effective antihypertensiveagents.

DETAILED DESCRIPTION OF THE INVENTION

The discovery of ACE inhibitory potency in the compounds of the presentinvention provides a unique approach to the design of inhibitorycompounds. Although many prior art inhibitors are proline derivatives,substitution of other amino acids for proline has also yielded potentinhibitors. Arginine, phenylalanine and alanine are all effectivesubstitutes for proline, so that a trend is not discernible.

The substitution of L-3,4-dehydroproline for proline has been studied inseveral systems. Substitution of L-3,4-ΔPro in the 7 position ofbradykinin yields a bradykinin derivative which has significantlyreduced physiological activity. See Fisher, G. H. et al., Arch. Biochem.Biophys. 189, 81 (1978). On the other hand, substitution of L-3,4-ΔProat the 3,5 or 9 position in ACE inhibitor BPP_(9a) enhances itsinhibitory activity. In copending application Ser. No. 958,180applicants found that the compounds having ΔPro, which are disclosed insaid application, have high inhibitory potency and antihypertensiveeffectiveness. However, at present, no rationale can be advanced toexplain the diversity of observed results following substitution of ΔProfor proline. Similarly, no clear picture has emerged of the effects ofother proline derivatives or analogs substituted at various loci on ACEinhibitors.

To date, the effect of the amino acid to the left of the sulfur in theabove-shown formula, has not been determined. It is thought that thisamino acid functions as an additional recognition site for the enzyme.If this is true, it would be expected that a compound with an amino acidhere would be a better inhibitor. It was not known which, if any, aminoacids would be effective in this position and which if any would enhancethe inhibitory activity of a given compound. Applicants have found thatvarious amino acids are effective and that the hydroxyprolines, proline,L-, and D,L-,3,4-dehydroproline, and thiazolidine-4-carboxylic acidderivatives are all effective anti-hypertensive agents and have highinhibitory potency for ACE.

The present invention will be further described by the followingexamples. In these examples, the thin-layer chromatography (TLC) wasperformed using silica gel plates. The numerical solvent systems for usein the TLC procedures are as follows.

(1) is methanol:chloroform, 1:1 (parts by volume). (2) isbenzene:water:acetic acid, 9:1:9 (parts by volume). (3) is aceticacid:water:n-butanol 26:24:150 (parts by volume). (4) isn-butanol:pyridine:acetic acid:water, 15:10:3:12 (parts by volume). (5)is chloroform:methanol:ammonium hydroxide, 60:45:20 (parts by volume).The buffers for paper electrophoresis were: pH 1.9--formic acid:aceticacid:water, 3:2:25 (parts by volume); pH 5.0--diethylene glycol:aceticacid:pyridine:water, 100:6:8.5:885 (parts by volume). Thetert-butyloxycarbonyl derivatives of the amino acids are commerciallyavailable.

EXAMPLE 1

ACE activity assay. For most experiments described herein, the enzymewas assayed in 0.05 M Hepes buffer, pH 8.0 containing 0.1 M NaCl and0.75 M Na₂ SO₄. The substrate employed was Benzoyl-GlyHisLeu at a finalconcentration of 1 ×10⁻⁴ M, (K_(m) ≈2×10⁻⁴ M), together with about130,000 cpm of [³ H]-Benzoyl-GlyHisLeu (25 Ci/mmole). Enzyme was dilutedin the above buffer such that 40 μl buffered enzyme was capable ofhydrolyzing 13% of substrate in a 15-minute incubation at 37° C. Toinitiate the assay, 40 μl of enzyme and 10 μl of water or inhibitordissolved in water were preincubated ror five minutes at 37° C.Substrate, 50 μl, was then added to initiate reaction and the solutionwas incubated for 15 minutes at 37° C. To terminate the reaction, 1 mlof 0.1 M HCl was added, following which 1 ml of ethyl acetate was added.The mixture was agitated on a rotary mixer and centrifuged briefly toseparate the phases.

An aliquot, 500 μl, of the ethyl acetate layer was transferred to aliquid scintillation vial containing 10 ml of Riafluor, trademark NewEngland Nuclear Corporation, Boston, Mass. For determination of I₅₀values, enzyme activity in the presence of inhibitor at a series ofdifferent concentrations was compared to activity in the absence ofinhibitor. A plot of inhibitor concentration versus percent inhibitionyielded the I₅₀ value.

EXAMPLE 2 Synthesis of 3-acetylthiopropanoyl-L-proline-t-butyl ester

3-acetylthiopropanoic acid, 0.865 g, was dissolved in 2 ml redistilledtetrahydrofuran (THF) and cooled to 0° C. A cooled solution ofdicyclohexylcarbodiimide, 1.2031 g in 2 ml of THF was added, followingwhich a cooled solution of L-proline-t-butyl ester, 1 g, was added. Thereaction mixture was stirred at 0° C. for one hour, then at 4° C.overnight. The reaction mixture was then filtered and the precipitatewas washed with ethyl acetate. Solvents of the filtrates were removedunder reduced pressure in a rotary evaporator. The residue was dissolvedin ethyl acetate which was then washed three times with cold 1 N citricacid, twice with saturated NaCl, twice with cold 1 N NaHCO₃ and threetimes with saturated NaCl. The solution was dried over anhydrous MgSO₄and filtered. The solvent was removed under reduced pressure in a rotaryevaporator at 30° C. yielding a clear colorless oily product inapproximately 87% yield. The product migrated as a single spot in thinlayer chromatography in five solvent systems.

EXAMPLE 3 Synthesis of 3-mercaptopropanoyl-L-proline

The product from Example 2, 3-acetylthiopropanoyl-L-proline-t-butylester, 0.5 g, was mixed with 4.5 ml of 5.5 N methanolic ammonia at roomtemperature under nitrogen for one hour to remove the acetyl group. Thesolvent was then removed at 25° C. with a rotary evaporator. After theproduct was taken up in methanol and reevaporated twice more in therotary evaporator, the clear oily residue was dissolved in ethyl ether,washed twice with 5% potassium bisulphate and once with saturated NaCl,dried over MgSO₄ and filtered. Residual solvent was removed in vacuo toyield a clear oily product, migrating as a single spot on thin layerchromatography in three separate solvent systems. The t-butyl esterprotecting group was removed by reaction with trifluoroacetic acid inanisole.

EXAMPLES 4-6

By substituting 2-acetylthiopropanoic acid,3-acetylthio-2-D-methylpropanoic acid, or3-acetylthio-2-D,L-methylpropanoic acid for the 3-acetylthiopropanoicacid in Example 2 and substantially following the procedures of Examples2 and 3, the following compounds are obtained. By removing the t-butylester protecting group with trifluoroacetic acid in anisole as a firststep, the dicyclohexylamine salt can be formed to assist in theresolution of isomers. The acetyl protecting group can be removed in asecond step using methanolic ammonia, as described in Example 3.

    ______________________________________                                        Example   Compound                                                            ______________________________________                                        4         2-mercaptopropanoyl-L-proline                                       5         3-mercapto-2-D-methylpropanoyl-L-proline                            6         3-mercapto-2-D,L-methylpropanoyl-L-proline                          ______________________________________                                    

EXAMPLE 7 Synthesis of3-mercapto-2-methyl-propanoyl-L-3,4-dehydroproline

L-3,4-dehydroproline (Δ³ Pro), 1 mmole, is dissolved in DMF and thesolution is cooled to -15° C. The solution is neutralized by adding 1equivalent of N-ethyl morpholine. In a separate reaction vessel at -10°C., one equivalent of 3-acetylthio-2-methyl-propanoic acid in an equalvolume of DMF is mixed with 1.1 equivalent of 1,1'-carbonyldiimidazole,and the solution is stirred for one hour. The first solution containingΔ³ Pro is mixed with the second, containing 3-acetylthio-2-methylpropanoic acid while maintaining the temperature at -10° C. The combinedsolution is stirred for 1 hour at -10° C. The solution is then allowedto warm slowly to room temperature. The solvent is removed on a rotaryevaporator under reduced pressure at 40° C. Ethyl acetate (25 ml) isadded and the solution is cooled to 0° C. Two ml of 1 N citric acid isadded, the two phases are mixed and then allowed to separate. The phasesare separated with a separating funnel, and the organic phase is washedtwice more with 2 ml 1 N citric acid, two times with saturated NaCl andfinally dried over anhydrous MgSO₄. The MgSO₄ is removed by filtration,and the solvent is removed with a rotary evaporator. The residue isdissolved and recrystallized from a non-polar solvent such as benzene toyield 3-acetylthio-2-D,L-methylpropanoyl-L-3,4-dehydroproline. When the2-D-methyl isomer is desired, the residue is dissolved in acetonitrile(approximately 3 ml) and the solution is warmed to 40° C. One equivalentof dicyclohexylamine is added, and the solution is allowed to stand atroom temperature overnight. The crystals are collected by filtration andare washed three times with acetonitrile. When further purification isrequired, the material can be recrystallized from isopropanol. Theacetyl protecting group can be removed as in Example 3.

EXAMPLES 8-11

By substituting D,L-3,4-dehydroproline, L-3-hydroxyproline,L-4-hydroxyproline, or L-thiazolidine-4-carboxylic acid for theL-3,4-dehydroproline in Example 7 and substantially following theprocedures of Example 7 the following compounds are obtained.

    ______________________________________                                        Example Compound                                                              ______________________________________                                         8      3-mercapto-2-D-methylpropanoyl-D,L-3,4-dehydro-                               proline                                                                9      3-mercapto-2-D-methylpropanoyl-L-3-hydroxyproline                     10      3-mercapto-2-D-methylpropanoyl-L-4-hydroxyproline                     11      3-mercapto-2-D-methylpropanoyl-L-thiazolidine-4-                              carboxylic acid                                                       ______________________________________                                    

EXAMPLE 12

Similarly, by substituting 3-acetylthiopropanoic acid or2-acetylthiopropanoic acid for the 3-acetylthio-2-methyl propanoic acidof Examples 7-11, the L-3,4-dehydroproline, D,L-3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline and L-thiazolidine-4-carboxylicacid derivatives are obtained, following substantially the describedprocedures.

EXAMPLE 13 Preparation of pyro-L-glutamyl-L-phenylalanine benzyl ester

A solution of 0.52 g of pyro-L-glutamic acid, 1.72 g of L-phenylalaninebenzyl ester toluenesulfonic acid and 0.55 mg of N-ethylmorpholine in 5ml of dimethylformamide (DMF) and 20 ml of dichloromethane was cooled inan ice bath with stirring. A solution of 0.826 g ofdicyclohexylcarbodiimide in 2 ml dichloromethane was added to the abovereaction mixture. The reaction mixture was stirred in an ice water bathfor 1 hour and then at room temperature overnight. Dicyclohexylurea wasremoved by filtration and the product was washed in ethyl acetate.Solvents of the combined filtrates were removed under reduced pressurewith a rotary evaporator at 40° C. Ethyl acetate (25 ml) was added tothe residue and the organic solution was washed until neutral. Theorganic phase was dried over anhydrous MgSO₄, filtered and then thesolvent was removed with a rotary evaporator. The material wascrystalized from isopropanol and ether, yield: 1.01 g of white needles,m.p. 103°-104.5° C. The material was homogeneous on all TLC andelectrophoresis systems.

Elemental analysis:

    ______________________________________                                                   Calculated                                                                            Found                                                      ______________________________________                                        C            68.84     68.58                                                  H            6.05      6.05                                                   N            7.64      7.56                                                   ______________________________________                                    

EXAMPLE 14 Preparation of pyro-L-glutamyl-L-phenylalanine

The benzoyl ester protecting group of the compound of Example 13 (1.0 g)was removed by catalytic hydrogenolysis with 150 mg of 10% (by weight)Pd on carbon in 0.15 ml of glacial acetic acid and 15 ml of ethanol at20 psi of H₂ at room temperature overnight. The catalyst was removed byfiltration. Solvent was removed with a rotary evaporator. The materialwas crystalized from isopropanol and benzene, to yield a total of 402 mgof white crystals, m.p. 147°-149° C. The material was homogeneous onelectrophoresis at pH 1.9 and pH 5.0 and on TLC in systems 1, 2 and 3.

Elemental analysis:

    ______________________________________                                                   Calculated                                                                            Found                                                      ______________________________________                                        C            60.86     60.37                                                  H             5.84     5.85                                                   N            10.14     9.98                                                   ______________________________________                                    

EXAMPLE 15 Preparation of N.sup.α -[3-(Pyro-L-glutamyl-L-phenylalanylthio)-2-D-methylpropanoyl]-L-proline

A solution of 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml DMF was addedto a solution of 139 mg of the product of Example 14 in 0.5 ml DMF at-15° C. The reaction mixture was stirred at -10° C. for 1 hour, and thena mixture of 119.5 mg of 3-mercapto-2-D-methylpropanoyl-L-proline and0.072 ml of N-ethyl morpholine in 1 ml. DMF was added. The reactionmixture was stirred at -10° C. for an additional hour and then wasslowly warmed to room temperature. DMF was removed under reducedpressure with a rotary evaporator at 40° C. and then 7 ml ethyl acetateand 2 ml 1 N citric acid were added. The organic phase was washed twotimes with 1 N citric acid and two times with saturated NaCl. Theorganic phase was dried with anhydrous MgSO₄ and then filtered. Solventwas removed using a rotary evaporator. The residue was purified onsephadex G-25¹ (1.2×99 cm) partition column chromatography withn-butanol: acetic acid: H₂ O (4:1:5 by volume). The product washomogeneous on TLC (systems 1, 2 and 3) and on electrophoresis at pH5.0.

EXAMPLE 16

By substituting N,.sup.α N.sup.ε -bis-t-butyloxycarbonyl-L-lysine(hereinafter bis-boc-L-Lys) or N.sup.α N.sup.γ, N.sup.δ-triadamantyloxycarbonyl-L-arginine (hereinafter tri-Adoc-L-arginine)for pyro-L-glutamic acid and by following substantially the procedure ofExample 13, the corresponding bis-boc-L-Lys or tri-Adoc-L-Argderivatives of L-Phe-benzyl ester will be synthesized. Bis-boc-L-Lys iscommercially available. Tri-Adoc-L-Arg is prepared according to Jager,G. and Geiger, R., Chem. Ber. 103, 1727 (1970).

By substituting the benzyl esters of Gly, Ala, Trp, Tyr, Ile, Leu, Hisor Val, in the procedures of Examples 13 and 16, the correspondingpyro-L-glutamyl, bis-boc-L-Lys and tri-Adoc-L-Arg derivatives areobtained. The foregoing benzyl esters are commercially available. Benzylester protecting groups are removed essentially as described in Example14.

EXAMPLE 17

The compounds resulting from Example 16 are reacted with any of thecompounds resulting from the procedures of Examples 4-12, followingessentially the procedures of Example 15, to yield the correspondingbis-boc-L-Lys and tri-Adoc-L-Arg derivatives. The bis-boc and tri-Adocprotecting groups are removed by treatment with trifluoroacetic acid inanisole.

Following essentially the procedures and methods of Examples 2-17, afamily of thiolester compounds is obtained which have the generalformula ##STR4## wherein, R is L-arginyl, L-lysyl or L-pyroglutamyl;

A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl,L-isoleucyl, L-leucyl, L-histidyl, or L-valyl;

R₁ is hydrogen or methyl;

R₂ is L-proline, L-3,4-dehydroproline, D,L-3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine-4-carboxylicacid whose imino group is in imide linkage with the ##STR5## and n is 0or 1 such as when n=0, R₁ is methyl.

EXAMPLE 18 Oral effectiveness of N.sup.α-[3-(pyro-L-glutamyl-L-phenylalanylthio)-2-D-methyl-propanoyl]-L-proline

Rats (210-290 g body weight) were fasted overnight and then anesthetizedwith intraperitoneal pentobarbital, 50-60 mg/kg. Tracheostomy wasperformed and the animals were ventilated mechanically. A cannula wasinserted into a femoral vein for injection of angiotensin I, and asecond cannula was inserted into a common carotid artery for directmeasurement of arterial blood pressure. Heparin, 1,000 units, wasinjected via the femoral vein to prevent coagulation. Blood pressure wasmeasured with a pressure transducer connected to a polygraph. The ratswere injected with 400 ng/ml of angiotensin I in 20 μl of 0.9 g % NaCl;an amount of angiotensin I sufficient to raise mean arterial bloodpressure by 38 mm Hg. After the responsiveness of a given rat toangiotensin I was established, the named compound at 20 μmol/kg (drugdissolved in 0.15 ml of H₂ O plus 10 μl of 1 N NaHCO₃), was given via astomach tube. At timed intervals, the effects of 400 ng/kg ofangiotensin I on mean arterial blood pressure were tested. Results areshown below:

    ______________________________________                                        Time After Oral Blood Pressure Response to                                    Administration  400 ng/kg of Angiotensin I                                    (Minutes)       (% of Control)                                                ______________________________________                                        -5              100        (38 mm Hg)                                         +1              53                                                             5              66                                                            15              26                                                            18              26                                                            21              21                                                            25              21                                                            29              24                                                            39              18                                                            44              16                                                            49              16                                                            54              18                                                            60              18                                                            74              18                                                            86              21                                                            110             21                                                            120             26                                                            132             26                                                            142             26                                                            ______________________________________                                    

EXAMPLE 19 Intravenous effectiveness of N.sup.α-[3-(pyro-L-glutamyl-L-phenylalanylthio)-2-D-methyl-propanoyl]-L-proline

Anesthetized rats were prepared as described in Example 18. After thecontrol reponse to 400 ng/kg angiotensin I was measured for a given rat,the named compound was administered via a femoral vein. The dose was 2μmol/kg of the named compound in 15 μl 0.01 N sodium bicarbonate. Theresults are shown as percent of the control response.

    ______________________________________                                                          Blood pressure response to                                  Time after intravenous                                                                          400 ng/kg angiotensin I                                     administration (minutes)                                                                        (% of control)                                              ______________________________________                                        -5                    100%      (38 mm Hg)                                    +1/2      (30 sec.)   8                                                       5                     18                                                      11                    21                                                      14                    24                                                      17                    29                                                      20                    34                                                      24                    42                                                      32                    47                                                      43                    53                                                      49                    58                                                      56                    61                                                      71                    71                                                      76                    68                                                      88                    76                                                      99                    76                                                      109                   74                                                      125                   84                                                      ______________________________________                                    

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A new compound having the formula ##STR6##wherein R is L-arginyl, L-lysyl or L-pyroglutamyl;A is L-phenylalanyl,glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl, L-leucyl,L-histidyl, or L-valyl whose amino group is in amide linkage with R; R₁is hydrogen or methyl; R₂ is a residue of L-proline,L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline,L-4-hydroxyproline or L-thiazolidine-4-carboxylic acid whose imino groupis in imide linkage with the adjacent ##STR7## and, n is 0 or 1, suchthat when n=0, R₁ is methyl.
 2. A compound of claim 1 wherein R₂ isL-proline.
 3. A compound of claim 1 wherein R₂ is L-3,4-dehydroproline.4. A compound of claim 1, 2 or 3 wherein R is L-pyroglutamyl, A isL-phenylalanyl, n is 1 and R₁ is methyl.
 5. A compound of claim 1, 2 or3 wherein A is L-phenylalanyl, L-tryptophyl, L-tyrosyl or L-histidyl. 6.A compound of claim 1, 2 or 3 wherein A is glycyl, L-alanyl,L-isoleucyl, L-leucyl or L-valyl.
 7. A method for inhibiting angiotensinconverting enzyme in vivo comprising administering an effective dose ofa compound having the formula ##STR8## wherein R is L-arginyl, L-lysylor L-pyroglutamyl;A is L-phenylalanyl, glycyl, L-alanyl, L-tryptophyl,L-tyrosyl, L-isoleucyl, L-leucyl, L-histidyl, or L-valyl whose aminogroup is in amide linkage with R; R₁ is hydrogen or methyl: R₂ is aresidue of L-proline, L-3,4-dehydroproline, D,L-3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline or L-thiazolidine4-carboxylicacid whose imino group is in imide linkage with the adjacent ##STR9##and, n is 0 or 1, such that when n=0, R₁ is methyl.
 8. The method ofclaim 7 wherein R₂ is L-proline.
 9. The method of claim 7 wherein R₂ isL-3,4-dehydroproline.
 10. The method of claim 7, 8 or 9 wherein R isL-pyroglutamyl, A is L-phenylalanyl, n is 1 and R₁ is methyl.
 11. Themethod of claim 7, 8 or 9 wherein A is L-phenylalanyl, L-tryptophyl,L-tyrosyl or L-histidyl.
 12. The method of claim 7, 8 or 9 wherein A isglycyl, L-alanyl, L-isoleucyl, L-leucyl, or L-valyl.
 13. A method forreducing blood pressure in vivo comprising administering an effectivedose of a compound having the formula ##STR10## wherein R is L-arginyl,L-lysyl or L-pyroglutamyl;A is L-phenylalanyl, glycyl, L-alanyl,L-tryptophyl, L-tyrosyl, L-isoleucyl, L-leucyl, L-histidyl, or L-valylwhose amino group is in amide linkage with R; R₁ is hydrogen or methyl;R₂ is a residue of L-proline, L-3,4-dehydroproline,D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline orL-thiazolidine-4-carboxylic acid whose imino group is in imide linkagewith the adjacent ##STR11## and, n is 0 or, such that when n=0, R₁ ismethyl.
 14. The method of claim 13 wherein R₂ is L-proline.
 15. Themethod of claim 13 wherein R₂ is L-3,4-dehydroproline.
 16. The method ofclaim 13, 14 or 15 wherein R is L-pyroglutamyl, A is L-phenylalanyl, nis 1 and R₁ is methyl.
 17. The method of claim 13, 14 or 15 wherein A isL-phenylalanyl, L-tryptophyl, L-tyrosyl or L-histidyl.
 18. The method ofclaim 13, 14 or 15 wherein A is glycyl, L-alanyl, L-isoleucyl, L-leucylor L-valyl.